ARTICLE

https://doi.org/10.1038/s41467-019-11726-4 OPEN and ubiquitin position Set2 to methylate H3K36

Silvija Bilokapic 1 & Mario Halic 1

Histone H3 lysine 36 (H3K36me) is a conserved modification deposited by the Set2 methyltransferases. Recent findings show that over-expression or mutation of Set2 enzymes promotes cancer progression, however, mechanisms of H3K36me are poorly

1234567890():,; understood. Set2 enzymes show spurious activity on and histone tails, and it is unknown how they obtain specificity to methylate H3K36 on the nucleosome. In this study, we present 3.8 Å cryo-EM structure of Set2 bound to the mimic of H2B ubiquitinated nucleosome. Our structure shows that Set2 makes extensive interactions with the H3 αN, the H3 tail, the H2A C-terminal tail and stabilizes DNA in the unwrapped conformation, which positions Set2 to specifically methylate H3K36. Moreover, we show that ubiquitin contributes to Set2 positioning on the nucleosome and stimulates the methyltransferase activity. Notably, our structure uncovers interfaces that can be targeted by small molecules for development of future cancer therapies.

1 Department of Structural Biology, St. Jude Children’s Research Hospital, 263 Danny Thomas Place, Memphis, TN 38105, USA. Correspondence and requests for materials should be addressed to S.B. (email: [email protected]) or to M.H. (email: [email protected])

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fi istone modi cations are a key player in maintaining Table 1 Cryo-EM data collection, refinement, and validation genome stability, chromosome segregation, and genome statistics H 1 fi expression . One of the essential histone modi cations, lysine 36 methylation (H3K36me) is deposited by the Set2_NCP Set2_Ub_NCP NCP EMD- Set2 methyltransferase onto the H3 tail and is conserved from 2,3 EMD-0559 EMD-20517 20516 PDB yeast to humans . Set2 is recruited by phosphorylated C- PDB PDB ID 6PX3 ID 6PX1 terminal domain of RNA Polymerase 2 during transcription and ID 6NZO 4–9 catalyzes H3K36 methylation over gene bodies . In addition to Data collection and processing Set2, RNA Polymerase 2 elongation complex recruits Bre1/Rad6 Magnification that ubiquitinate H2B K12010,11, indicating possible interplay Voltage (kV) 300 300 300 between these two histone modifications. Both, H3K36me and Electron exposure 75 75 75 H2B K120 ubiquitination are enriched over gene bodies12. (e–/Å2) H3K36me recruits Rpd3S histone deacetylase complex to restore Defocus range (μm) −0.7 to −3.0 −0.7 to −3.0 − 0.7 to −3.0 a repressed state behind the elongating Pixel size (Å) 1.04 1.04 1.04 13,14 Symmetry imposed C1 C1 C2 polymerase . Although deposited by the transcription ∼ ∼ ∼ machinery, H3K36me seems to be a silencing mark that prevents Initial particle 1,500,000 1,500,000 1,500,000 2,15–17 images (no.) initiation of cryptic transcription within gene bodies . Final particle 66,000 50,000 210,000 Recent data show that Set2 and H3K36me are also involved in images (no.) 18–20 21,22 DNA damage repair , regulation of pre-mRNA splicing , Map resolution (Å) 3.8 4.1 3.3 chromatin condensation23, and histone exchange24,25. FSC threshold In animals, Set2 family enzymes are essential for maintenance Map resolution 3.6–5.0 3.8–8.0 3.1–3.5 of cell identity26,27 and are mutated in many patients with clear range (Å) cell renal cell carcinoma, acute leukemias, bladder cancer, and Refinement glioblastoma28–30. Recently, mutation of H3.3K36 to methionine Initial model used 6FQ5 6FQ5 6FQ5 has been found in chondroblastomas, head and neck squamous Model resolution (Å) 3.8 4.1 3.3 31–36 FSC threshold cell carcinoma, and colorectal cancer . H3.3K36M mutation Model resolution 235–3.8 235–4.1 235–3.3 traps SETD2 methyltransferase on the substrate which results in range (Å) genome-wide loss of H3K36 methylation and tumor Map sharpening B −100 0 −120 formation33,35. Set2 family enzymes are overexpressed in multiple factor (Å2) myeloma, neuroblastoma, bladder and breast cancers, and cor- Model composition relate with poor prognosis3,37–40. These findings show that 13740 14336 11939 overexpression or mutation of Set2 enzymes or H3K36 promote Nonhydrogen atoms cancer progression. Protein residues 990 1060 752 Ligands 3 3 0 Although Set2 involvement in essential cellular processes is 2 well described, mechanisms of Set2 mediated H3K36 methylation B factors (Å ) Protein 55.16 297.17 41.58 are poorly understood. In this study, we present 3.8 Å cryo-EM Ligand 129.02 397.28 structure of Set2 bound to the mimic of H2B ubiquitinated R.m.s. deviations 41 nucleosome core particle (NCP) . Our structure shows that Set2 Bond lengths (Å) 0.007 0.008 0.006 makes extensive interactions with H3 αN, H3 tail, H2A C- Bond angles (°) 0.914 0.929 0.853 terminal tail, and stabilizes DNA in an unwrapped conforma- Validation tion42. These interactions with the nucleosome are required for MolProbity score 1.47 1.55 1.01 the specificity toward H3K36 methylation. Moreover, we show Clashscore 3.44 4.62 2.31 that the AWS domain of Set2 interacts with ubiquitin which Poor rotamers (%) 0.12 0.45 0 promotes H3K36 methylation. Our structure reveals the interface Ramachandran plot Favored (%) 95.11 95.45 98.10 between nucleosome and Set2, which is a potential drug target for Allowed (%) 4.89 4.55 1.90 cancer treatments. Disallowed (%) 0.0 0.0 0.0

Results Cryo-EM structure of Set2 bound to nucleosome. Histone resolved to high resolution allowing us to describe interactions in H3K36me3 and H2BK120 ubiquitination are hallmarks of active molecular details. In this map, resolution of Set2 on the nucleo- chromatin. To visualize the mechanism of H3K36 methylation, some proximal side is similar to the nucleosome with many side we assembled the complex of Set2 and the mimic of H2B ubi- chains resolved, whereas on the nucleosome distal side the quitinated nucleosome43, the substrate of Set2 (Supplementary resolution drops to ~4–5 Å (Supplementary Fig. 3b-c). Although Fig. 1a-d). To stabilize the complex we have introduced H3K36M we used full-length Set2 for the complex formation, in our map mutation44,45, recently identified in childhood cancers (Supple- we observe only densities for the AWS and the SET domains mentary Fig. 1e). In the electron micrographs the complex is (Supplementary Fig. 3d). This is in agreement with previous data present in various orientations and in a subset of 2D class showing that the SET domain is sufficient to bind nucleosome averages an additional density was bound to the NCP (Supple- and to methylate H3K366,46,47.Werefined the cryo-EM model of mentary Fig. 1f, g). In initial 3D reconstruction, with overall the nucleosome assembled on 601 DNA42,48 (PDB:6FQ5) and the resolution at 3.4 Å, a noisy density representing Set2 domain was Set2 model (PDB:5V21, 5JJY)44,45 and observed several changes present near the DNA entry/exit site of the nucleosome (Sup- in their structures when assembled in the complex (Fig. 1b and plementary Fig. 2). ~14% of the initial particles contained Set2 Supplementary Fig. 3e). and further focused classification improved the Set2 density Our cryo-EM structure shows that Set2 stabilizes the yielding a map at 3.8 Å (Set2:NCP) (Table 1, Fig. 1a and Sup- unwrapped state of the nucleosome, which resembles the Class plementary Fig. 2, 3a). Remarkably, not only NCP, but also Set2 is 2 state we have previously observed42 (Fig. 1a, b). The DNA

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abSet2:NCP complex Set2:NCP complex DNA entry/exit site Unwrapped DNA

Set2 Set2

Dyad Dyad

Nucleosome Nucleosome

Fig. 1 Cryo EM structure of Set2 methyltransferase bound to the NCP. a Cryo-EM map of Set2 bound to NCP at 3.8 Å. NCP is shown in gray and Set2 in pink. Set2 stabilizes NCP with the unwrapped DNA. b Model for cryo-EM map of the Set2 bound to NCP. NCP is shown in gray and Set2 in pink

ab DNA (SHL 5.5) Set2 Set2 DNA (SHL-1) K241

H3 tail R117 K322

T323

c d H3 N-term tail Set2 Set2 Set2 Set2 K37 N295 DNA N151 D125 (SHL-1) H39 R40 T154 K56 K269 R52 R49 T45

Y41 K155 H3 αN e Set2 Set2 D146

I150 A153 I111 K119 L116

H2A C-term tail

Fig. 2 Molecular interactions between Set2 and NCP. Global location of the depicted interaction between Set2 and the nucleosome is shown on the left and close up on the right. a Set2 residue R117 binds the backbone of the unwrapped DNA at SHL 5.5. b Set2 residues K241, K322, and T323 bind the backbone of the DNA at SHL-1. c Set2 binds H3 αN. Set2 residues D125, N151, and T154 bind the H3 K56, R52, R49 and T45. d The H3 tail makes extensive interactions with Set2 and the DNA. H3 R40 and K37 bind the DNA at SHL-1. H3 Y41 and H39 interact with Set2 residues K155, K268, and N295. Set2 residue K269 interacts with the main chain of H39. e Set2 binds the H2A C-terminal tail. Set2 D146 makes charged interaction with H2A K119. Set2 residues I150 and A153 make hydrophobic interactions with the H2A residues I111 and L116, respectively unwrapping is required for Set2 to bind H3K36 that is located functional interaction with H3K36. Set2 residues D125 and N151 close to the DNA entry/exit site of the nucleosome. The interact with positively charged residues K56 and R52, while H3 conserved positively charged Set2 residue R117 binds the R49 and T45 face toward the Set2 A153 and T154 (Fig. 2c). These backbone of the unwrapped DNA at SHL 5.5 and stabilizes the H3 αN residues organize the last 13 bp of DNA in the open conformation (Fig. 2a and Supplementary Fig. 3e, f). nucleosome, but as DNA is unwrapped in the complex, the side Moreover, Set2 makes a set of electrostatic interactions with the chains adopt different conformation and bind Set2 (Supplemen- backbone of the DNA near the dyad (Fig. 2b and Supplementary tary Fig. 3c, i). Set2 interaction further extends to the H3 tail Fig. 3g). This interaction with the DNA at SHL-1 rearranges the where H3 Y41 binds into the pocket formed by H3 residues R49, Set2 Post-Set domain when compared with the crystal structure44 V46, and T45, and by Set2 residues K155 and K269. Y41 aromatic (Supplementary Fig. 3h). ring packs against invariant Set2 K269, which together with N295 In addition to the DNA, Set2 makes multiple interaction with and N297 also coordinates H3 H39 (Fig. 2d and Supplementary histones H3 and H2A that position Set2 catalytic domain for Fig. 4a). When H3 tail is bound to Set2, positively charged

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L48 (Fig. 2e). In addition to prevailing charged interactions, our a Unwrapped b Set2 DNA structure shows that Set2 binds hydrophobic patch of the α I150 nucleosome formed by the H2A C-terminal tail and the H3 N A153 (Fig. 3a, b and Supplementary Fig. 4e). This hydrophobic patch, H3 tail utilized by Set2 is likely to be another hot spot for interactions H3 αN 50 L48 with the nucleosome, similarly to the acidic patch . Biochemical Hydrophobic data have shown that Sgo1, COC complex, AKT protein kinase B, patch ISWI remodelers, and many other complexes require residues in I51 L116 the hydrophobic patch for nucleosome binding and the I111 51–53 NCP L115 activity and might bind the nucleosome in a similar way to H2A C-tail V114 Set2. Notably, we do not observe Set2 SET domain binding to the acidic patch. c We observed that variant H2A.B does not have 100 110 120 the hydrophobic residues that interact with Set2 (Fig. 3c and H2A1 . B H2A1 . A Supplementary Fig. 5a, b). H2A.B histone variant is associated H2A . X with active transcription54,55, however, in contrast to the H2A . Z canonical H2A, in vivo data show that H2A.B containing MacroH2A . 1 55 H2A . B are not methylated on H3K36 . Residues essential for Set2 binding are not present in H2A.B nucleosomes and our results explain why these nucleosomes do not have H3K36me, d although found in regions that are methylated on H3K36. Our DNA K241 R220 R222 DNA data provide insight into the modulation of chromatin landscape R117 K322 fi K225 K228 K269 by interplay between histone post-translational modi cations and K249 histone variants. In our structure Set2 makes extensive charged and hydro- 180° R117 phobic interactions with the DNA and the histones. The structure shows that Set2 residues facing the DNA are predominately D126 positively charged, whereas, the residues that are facing the D128 positively charged histones are negatively charged (Fig. 3d). D130 Notably, the residues interacting with the nucleosome show E133 D128 D162 higher degree of conservation (Supplementary Fig. 5c). E133 D137 D134 DNA Histones E167 Histones

Fig. 3 Set2 makes hydrophobic and charged interactions with NCP. H2BK120 ubiquitination stimulates Set2 activity. Set2 is a Hydrophobic residues on the surface of NCP are shown in violet. Black recruited to chromatin by RNA Polymerase II elongation com- dashed circle marks hydrophobic patch on the NCP. b Hydrophobic patch of plex, which also recruits Bre1 E3 ligase that ubiquitinates H2B. NCP is formed by H3 αN residues I51 and L48 and H2A C-terminal tail Moreover, recent in vivo data reveal a link between H2B ubi- 41 residues I111, L115, and L116. Set2 residues I150 and A153 bind the quitination and H3K36me . Therefore, we have postulated that hydrophobic patch of NCP. c Sequence alignment of the C-terminal tail of H2B ubiquitination might stabilize the Set2:NCP complex for the representative H2A histone variants. Conserved residues are cryo-EM studies. In the Set2 map, the density for ubiquitin is fl highlighted in red. The position of I111 and L116, residues that build visible only at low contour level, indicating local exibility fi hydrophobic pocket on the nucleosomal surface, are labeled with the red (Supplementary Fig. 6a, b). We have used focused classi cation to cross. V114 and L115 that position H2A C-terminal tail are marked with blue improve the ubiquitin density, which yielded maps with no visible fl fi triangle. Residues important for the interactions with Set2 enzyme are not ubiquitin, exible ubiquitin, and de ned ubiquitin (Supplemen- conserved in H2A.B. The alignment was generated with the ESPript server tary Fig. 6c). In all maps, Set2 was in the same conformation, (http://espript.ibcp.fr)83. d Electrostatic surface potential of Set2. indicating that H3K36M mutation is the primarily stabilizing Set2 surface facing DNA is predominately positively charged (blue), factor in our construct. In the cryo-EM map (Set2:Ub_NCP) at – whereas Set2 residues facing positively charged histones are mainly 4.1 Å ubiquitin is resolved to 5 8 Å and secondary structure fi negatively charged (red) elements are visible permitting us to t the ubiquitin crystal structure (PDB:1UBQ) (Fig. 4a and Supplementary Fig. 6d-g). In our complex, Set2 residues 129–135 of the AWS domain come to residues H3 R40 and K37 face toward the DNA and contribute to proximity to residues 63–67 of the C-terminal β-strand of ubi- position the H3 tail for the interaction with Set2 (Fig. 2d). quitin (Fig. 4b and Supplementary Fig. 6e). Whereas H3 M36(K36) is positioned in a similar way to Set2 Although this interaction is not well defined in the structure, crystal structures44,45, the H3 residues following the H3 P38 take our data suggest that ubiquitin helps positioning Set2 on the a different path in our structure and bind both Set2 and the nucleosome. Therefore, we have generated ubiquitinated nucleo- nucleosomal DNA (Supplementary Fig. 4b, c). It has been somes by ligating ubiquitin to H2B K12056 (Supplementary suggested that P38 plays a role in the H3K36 methylation49 and Fig. 7a-f) and by fusing ubiquitin to the H2A N-terminus (R17), our data show that the H3 tail kinks at the P38 to accommodate which is an efficient mimic of naturally occurring H2BK120 restrains provided by the nucleosome and to allow for Set2 ubiquitination43,57 (Supplementary Fig. 1b-d and Supplementary binding. Fig. 7g). Our data show that Set2 activity is higher on both Beside the interactions with H3, Set2 residue D146 makes an mimics of H2B K120 ubiquitinated nucleosomes when compared interaction with K119 in the H2A C-terminal tail and rearranges with the unmodified nucleosomes, indicating that ubiquitin the tail (Fig. 2e and Supplementary Fig. 4d). Moreover, Set2 stimulates the activity (Fig. 4c, d and Supplementary Fig. 8a, b). residues I150 and A153 make hydrophobic interactions with the Further, we added Set2 to an equimolar mix of unmodified and H2A C-terminal tail I111 and L116, and H3 αN residues I51 and H2B ubiquitinated nucleosomes, and observed that Set2

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a Set2:Ub_NCP complexbe Set2:Ub_NCP complex

20 40 60 0 Ubiquitin Ub_NCP Set2 (H2BK120C ligated) NCP αH3K36me3 Ub_NCP Dyad (H2BK120C ligated) Ubiquitin Set2 NCP Input (DNA) Residues 63–67 Residues 129–135

c d 100

80 0 3 6 12 24 0 3 6 12 24 Min 60 αH3K36me3

activity 40 αH4 20 H3K36 methylation Set2 0 0 51015 20 25 NCP Ub_NCP (H2BK120C ligated) Time H3K36me3 (Ub_NCP H2BK120C ligated) H3K36me3 (NCP)

Fig. 4 Ubiquitin binds Set2 and stimulates Set2 H3K36me3 activity. a Cryo-EM map resolved to 4.1 Å showing a subset of particles with defined density for ubiquitin, which interacts with Set2. Ubiquitin density and model are shown in violet. Set2 density and model are shown in pink. NCP is shown as model in gray. b The model showing overall interaction between Set2 and ubiquitin. Ubiquitin residues 63–67 come to close proximity to Set2 AWS domain (residues 129–135). c Set2 methyltransferase assay with the unmodified and the H2BK120C ubiquitinated nucleosomes. In presence of H2BK120C ubiquitin, Set2 shows increased H3K36me3 when compared with unmodified nucleosome. d Quantification of Set2 methyltransferase activity assay from (c) . Error bars show standard error of three independent replicates. e Set2 methyltransferase competition assay with equimolar amounts of both the unmodified and the H2BK120C ubiquitinated nucleosomes. The nucleosomes were separated on the native gel: sample stained with SybrGold is shown in the lower panel and tri-methylation activity detected with H3K36me3 antibody in the upper panel. Upper band is the H2BK120C ubiquitinated nucleosomes and the lower band are the unmodified nucleosomes. Source data are provided as a Source Data file

Unwrapped DNA Set2 SET domain Set2 disordered region Ubiquitin Set2 disordered region

Ubiquitin 2 Unwrapped DNA

Fig. 5 Set2 auxiliary regions bind the nucleosome. Cryo-EM map resolved to 4.2 Å showing a subset of particles with the noisy density next to the nucleosome. The noisy density is spanning from the Set2 SET domain and the unwrapped DNA to the second ubiquitin on the opposite side of the nucleosome. This density is generated by auxiliary Set2 domains and disordered regions. Ubiquitin is shown in violet, Set2 in pink, and NCP in gray preferably methylates H2B ubiquitinated nucleosomes (Fig. 4e). two Set2 bound. We have, however, observed a cluster of a noisy Although H2B K120 ubiquitin increases Set2 activity, it does not density on the side of the nucleosome opposite to the bound Set2 increase Set2 binding to the nucleosome, suggesting that SET domain (Fig. 5). Further unfocused classification has enri- ubiquitin rather promotes positioning of the Set2 active site over ched for this noisy density generated by the remaining the substrate (Supplementary Fig. 8c). In conclusion, our data Set2 sequence, which includes several domains and disordered show that H2B K120 ubiquitination facilitates H3K36me3 activity regions (Supplementary Figs. 3d, 9a-b). This less defined parts of of Set2. Set2 interact with the Set2 SET domain, unwrapped DNA, ubi- quitin and the histone core at multiple sites (Fig. 5). Our data reveal that the auxiliary domains and disordered regions of Set2 Auxiliary domains of Set2 bind nucleosome. Although two Set2 bind the nucleosome and ubiquitin, and this prevents binding of could bind one nucleosome, we did not find a single class with the the second Set2 to the nucleosome (Fig. 5).

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Discussion nucleosomes compared with the isolated SET domain70, indi- In conclusion, our data show that Set2 interactions with the H3 cating that auxiliary domains promote and modulate H3K36 αN and the H2A C-terminal tail position Set2 on the nucleosome methylation. to specifically bind and methylate H3K36. In agreement with our Perturbations in the enzymes that maintain the levels of structure, previous work has shown that hydrophobic residues H3K36 methylation have been reported in many diseases, such as I111, L115, and L116 in the H2A C-terminal tail are required for Sotos syndrome, acute myeloid leukemia, multiple myeloma, and the H3K36 methylation and repression of cryptic prostate cancer. Set2 enzyme NSD2 is overexpressed in multiple transcription58,59. Consistently, mutations in H2A residues resi- myeloma, bladder, and several other cancers, which correlates dues Q114 and N115, that do not bind Set2 in our structure, with poor prognosis37,38,71, making these enzymes potential drug showed no or little effect on H3K36me58. Notably, ubiquitination targets. The interaction with the nucleosome we describe in this of H2AK119 was shown to inhibit Set2 activity, which is con- study could be targeted by small molecules to inhibit Set2 sistent with our structure in which Set2 makes extensive inter- enzymes in treatment of various cancers. actions with this part of the H2A tail60. Mutational analysis has shown that residues T45, R49, and R52 Methods in H3 αN are required for H3K36 methylation and repression of Chaetomium thermophilum genomic DNA preparation. Twenty milliliters of cryptic transcription58,59,61,62, in agreement with our structure. saturated C. thermophilum culture were harvested by centrifugation. Cells were fl Previous work has shown that H4K44 is required for Set2 resuspended in 0.5 ml of dH2O and ash frozen as small droplets in liquid 58,61 nitrogen. Cells were pulverized under dry ice by mortar and pestle, thawed, and activity . Although we do not see direct interaction of H4K44 resuspended in extraction buffer (2 ml of 2% Triton X-100, 1% SDS, 100 mM NaCl, with the Set2 SET domain, our data show that H4K44 organizes 10 mM Tris/HCl, pH 8.0, 1 mM EDTA). In total, 1.5 ml of phenol:chloroform: the H2A C-terminal tail and the hydrophobic patch that are isoamyl alcohol (25:24:1) and glass beads were added to pulverized cells. The essential for Set2 activity. mixture was vigorously mixed by vortex for 5 min and aqueous layer was isolated by centrifugation. DNA was precipitated using sodium acetate (final concentration The Set2:nucleosome complex is further stabilized by the 0.3 M, pH 5.2) and 2.5 volumes of ice-cold ethanol, collected by centrifugation and interaction with the DNA, which is consistent with observations redissolved in TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA). that DNA is required for Set2 activity61,63. Moreover, in absence of the intact nucleosome Set2 family enzymes show spurious Plasmid construction. Chaetomium thermophilum (source Ed Hurt lab) Set2 was activity toward H3K4, H3K27, H4K20, H4K44, H2A, and H2B amplified from the genomic DNA and cloned into a pET-Duet plasmid to yield an residues3,63. Our structures explain how interaction with the N-terminally 6xHis-tagged protein followed by a human rhino-virus 3C protease fi recognition site. Two intrones contained within the genomic DNA were removed nucleosome provides the speci city to H3K36 methylation. by iPCR72. Oligonucleotides used for cloning are listed in Supplementary Table 1. Our data show that the Set2 AWS domain interacts with the The resulting vector, listed in Supplementary Table 2, was transformed into ubiquitin on H2BK120, suggesting that ubiquitin helps posi- LOBSTR-BL21(DE3)-RIL (Kerafast)73. tioning Set2 on the nucleosome. This is supported by our bio- Xenopus laevis histone H3K36M and H2BK120C mutant plasmids were generated by iPCR using oligonucleotides listed in Supplementary Table 1. The chemical work that shows increased Set2 tri-methylation activity resulting vector was transformed into BL21(DE3)-pLysS bacterial cell. on H2BK120 ubiquitinated nucleosomes, when compared with Ubiquitin fused to the N-terminus of H2A is an efficient mimic of naturally the unmodified nucleosomes. Moreover, it is consistent with the occurring H2BK120 ubiquitination43. Gene coding for S. cerevisiae ubiquitin was in vivo data showing reduced H3K36me3 in deletion of the Bre1 PCR amplified from genomic DNA and cloned into NcoI recognition sequence in 43 E3 ligase43, which is required for H2B K120 ubiquitination. the plasmid for X. laevis coexpression of soluble H2A/H2B histone dimer . Restriction enzyme cloning positioned ubiquitin gene between region coding for Previous biochemical and in vivo data also noted the considerable 6xHis-tag and H2A. A short HA tag used for tethering ubiquitin to H2A43 was spatial plasticity in the localization of the residue that is ubiqui- replaced in our construct with an equally long Gly-Ser linker by iPCR. The final tinated for nucleosome methylation by Dot1 enzyme. Ubiquitin plasmid construct contained N-terminal 6xHis-tag followed by thrombin ligated to H2BK120, H2BK125, H2AK22, and fused to recognition site, ubiquitin and nine residue Gly-Ser linker tethered to H2A R17. Oligonucleotides used are listed in Supplementary Table 1. H2AS17 stimulated Dot1 activity43,57,64. In agreement, our data show that ubiquitin ligated to H2BK120 and fused to Inverse PCR. iPCR reactions were setup in a total volume of 25 μl. Products were H2AR19 stimulates Set2 activity. Since ubiquitin is linked to the analyzed on the agarose gel and purified using the NucleoSpin Gel and PCR Clean- nucleosome by intrinsically flexible C-terminal tail, it can adopt up kit. Ten microliters of purified PCR product were incubated with 1 × T4 DNA many positions on the nucleosome. Remarkably, the position of ligase buffer and 5 U of T4 PNK for 1 h at 37 °C in a total volume of 20 μl. T4 DNA ubiquitin on the nucleosome surface is unique in each of several ligase (200 U) was added to the reaction and incubated for 1 h at room tempera- ture. After addition of 10 U of DpnI, the reaction was incubated for 1 h at 37 °C recently published structures of various factors bound to the and 5 μl were transformed into competent XL1-Blue E. coli cells. All obtained 65–67 H2BK120 ubiquitinated nucleosomes (Supplementary plasmid sequences were checked by DNA sequencing. Fig. 9c). This indicates that binding to the interacting partner determines position of the flexible ubiquitin on the nucleosome. Set2 expression and purification. E. coli Rosetta (Novagen) bacterial culture, Recently, a structure of H3K36 methyltransferase ASH1L was containing vector for Set2 overexpression, was grown in LB medium containing the determined in a complex with its activation partner MRG1568,69. appropriate antibiotics and 0.4% (w/v) glucose at 37 °C. When the optical density reached 0.6 at 600 nm, the culture was shifted to 18 °C for 30 min before it was Superposition of the SET domains from ASH1L:MRG15 com- induced by the addition of 0.2 mM IPTG. After overnight induction at 18 °C, cells plexes (PDB ID: 6ago, 6ine) and Set2:NCP complex shows that were harvested by centrifugation, resuspended in lysis buffer (50 mM sodium- different interfaces of Set2 bind nucleosome and MRG5 (Sup- phosphate, pH 8.0, 500 mM NaCl, 20 mM imidazole, 3 mM β-mercaptoethanol, 1 plementary Fig. 9d). This indicates that an activating factor mM phenylmethanesulfonyl fluoride and Benzonase) and lysed using a french MRG5 can bind Set2 that is bound to the nucleosome to further press. The cleared supernatant was incubated with Ni Sepharose 6 Fast Flow resin for 30 min at 4 °C. The resin was extensively washed with lysis buffer in batch and stimulate its activity. loaded onto a disposable column. On the column, the resin was washed with 4 bed In our structure we observed a cluster of less defined density volumes of washing buffer (50 mM sodium-phosphate, pH 8.0, 500 mM NaCl, 40 that spans from the SET domain and unwrapped DNA to the mM imidazole, 3 mM β-mercaptoethanol) and the bound protein was eluted with 4 H2A/H2B on the opposite side of the nucleosome. Our structure bed volumes of elution buffer (50 mM sodium-phosphate, pH 8.0, 500 mM NaCl, 300 mM imidazole, 3 mM β-mercaptoethanol). The protein was dialyzed overnight suggest that remaining Set2 domains and/or disordered density at 4 °C against 20 mM HEPES/NaOH, pH 7.5, 200 mM NaCl, 0.1 mM EDTA, 1 bind the SET domain, ubiquitin, unwrapped DNA, and histone mM DTT. The N-terminal tag was proteolytically removed during dialysis. The core at multiple sites. This is in agreement with biochemical data protein was further purified on size exclusion Superdex 200 Increase 10/300 showing that full-length Set2 shows higher activity on equilibrated in 20 mM HEPES/NaOH, pH 7.5, 200 mM NaCl, 1 mM DTT. All

6 NATURE COMMUNICATIONS | (2019) 10:3795 | https://doi.org/10.1038/s41467-019-11726-4 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11726-4 ARTICLE protein purification step were done at 4 °C. The fractions with the protein were mM imidazole, 3 mM β-mercaptoethanol). The eluted protein mixture was directly concentrated, flash frozen and stored at −80 °C as single use aliquots (Supple- used in the subsequent refolding steps with H2A histone (Supplementary Fig. 7b). mentary Fig. 1a). Reconstitution of histone dimers with H2BK120C mimic. H2A histone, for fi dimer reconstitution, was overexpressed in BL21(DE3) pLysS cell strain and pur- Histone expression and puri cation. The plasmid for coexpression and copur- fi 74,75 fi i ed from the inclusion bodies . To remove minor impurities, H2A histone was i cation of X. laevis histone pairs (6xHis_HRV3C_H2A/H2B, 6xHis_Ubq_H2A/ further purified on cation-exchange chromatography under denaturating condi- H2B, 6xHis_HRV3C_H3/H4) were transformed into E. coli Rosetta cells (Sup- fi fi tions as described. The puri ed H2A was dialyzed thoroughly against at least four plementary Table 2). Protein expressions and Ni-NTA puri cations were done as changes of distilled water at 4 °C and lyophilized. Lyophilized protein was resus- described for Set2 methyltransferase enzyme except that all buffers contained 2 M pended in unfolding buffer (6 M guanidinium hydrochloride, 20 mM Tris, pH 7.5, NaCl. After the elution from the resin, the histone pairs were dialyzed overnight at 1 mM DTT), allowed to unfold for 60 min at room temperature and spun by 4 °C against 25 mM HEPES/NaOH, pH 7.5, 2 M NaCl, 0.1 mM EDTA, 1 mM DTT. fi centrifugation for 20 min at 17,000 rpm at 4 °C. The samples were further puri ed via cation-exchange chromatography in batch The nickel affinity purification eluate of nonhydrolyzable ubiquitin-histone (SP Sepharose Fast Flow resin). To facilitate binding onto the resin, the samples contains a mixture of ubiquitin-containing species: his-tagged ubiquitin, his-tagged were diluted with buffer containing 25 mM HEPES/NaOH, pH 7.5 and 1 mM DTT ubiquitin-ubiquitin, and his-tagged ubiguitin-H2B. Thus, the ratio of H2A and to 500 mM salt concentration. The resin was extensively washed with 500 mM salt ubiquitin-H2BK120C histone mimic for dimer reconstitution was estimated by buffer in batch and loaded onto a disposable column. On the column, the resin was SDS-PAGE and staining with SimplyBlue SafeStain. Histones were mixed at equal washed with 2 bed volumes of buffer containing 700 mM salt (25 mM HEPES/ molar ratio, dialyzed against two changes of refolding buffer (25 mM HEPES/ NaOH, pH 7.5, 700 mM NaCl and 1 mM DTT). The samples were eluted with 4 NaOH, pH 7.5, 2 M NaCl, 1 mM DTT) at 4 °C. Histone dimer was concentrated bed volumes of 25 mM HEPES/NaOH, pH 7.5, 2 M NaCl, 1 mM DTT. Histone fi fi and further puri ed on a size exclusion chromatography Superdex 200 Increase 10/ proteins were concentrated and further puri ed on the size exclusion Superdex 200 30 column, pre-equilibrated in buffer containing 15 mM HEPES/NaOH pH 7.5, 2 Increase 10/300 GL column equilibrated in 25 mM HEPES/NaOH, pH 7.5, 2 M M NaCl, 1 mM DTT. The cleanest fractions were pooled, concentrated and used NaCl, 1 mM DTT. The fractions with the protein were analyzed on SDS-PAGE, for histone octamer assembly (Supplementary Fig. 7c-f). concetrated and flash frozen. H3K36M and H3K120C mutants were overexpressed in BL21(DE3) pLysS cell strain and purified from the inclusion bodies74. Pelleted cells were resuspended in Nucleosome reconstitution. Nucleosomes were reconstituted using the purified wash buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT) octamers and 149 bp DNA fragment containing the 601 nucleosome strong posi- and sonicated. After centrifugation for 20 min at 4 °C and 17,000 rpm the pellet tioning sequence42,48. DNA for nucleosome reconstitution was PCR amplified from was resuspended in wash buffer with 1% (v/v) Triton X-100, sonicated once more a plasmid containing the 601 DNA sequence using oligonucleotides listed in and spun down. The pellet containing inclusion bodies of the corresponding Supplementary Table 1. After ethanol precipitation, the DNA was resuspended in histone protein was washed by completely resuspending it once in buffer with 15 mM HEPES-NaOH pH 7.5, 2 M NaCl, 1 mM DTT. Triton X-100 and twice in buffer without Triton X-100. After each resuspension, The nucleosome reconstitution was done by ‘double bag’ dialysis method48. The the sample was spun for 20 min at 4 °C and 17,000 rpm. H2BK120C histone was dialysis buttons, containing 0.25 ml of histone octamer:DNA mixture, were placed further purified on cation-exchange chromatography under denaturing conditions. inside a dialysis bag filled with 50 ml 2 M salt buffer (15 mM HEPES-NaOH pH Shortly, the histone protein was disolved in 7 M urea, 50 mM Tris-Hcl, 400 mM 7.5, 2 M NaCl, 1 mM DTT). The dialysis bag was dialyzed overnight against 1 l of 1 NaCl, 1 mM DTT. Any insoluble components were removed by centrifugation. The M salt buffer (15 mM HEPES-NaOH pH 7.5, 1 M NaCl, 1 mM DTT). The next day protein was bound to cation-exchange column and eluted with the increasing salt the dialysis bag was dialyzed for 5–6 h against 1 l buffer containing 15 mM HEPES- gradient. After elution from the ion exchange column, the protein was extensively NaOH pH 7.5, 50 mM NaCl, 1 mM DTT. Finally, only the dialysis buttons were dialyzed against water, lyophilized and used in cross-linking reaction with dialyzed for 1 h in fresh 50 mM salt buffer. All nucleosome reconstitution steps ubiquitin. H3K36M was refolded with H4, also purified from the inclusion bodies, were done at +4 °C. The reconstitution results were analyzed on 6% native PAGE into histone tetramer. Tetramer was further purified under native conditions on run in 1X TBE buffer at 200 V in the cold room (Supplementary Fig 1c). The gels ion exchange and size exclusion chromatography using protocol described above were stained with SYBR gold (Thermo Fisher Scientific). for co-expressed soluble histone pairs. To obtain the histone octamer, we mixed individually purified H2A/H2B CryoEM grid preparation and data collection histone dimer (H2A/H2B, Ubq_H2A/H2B or H2A/H2BK120C_Ubq) and (H3/H4) . The formation of Set2:NCP 2 histone tetramer ((H3/H4)2 or (H3K36M/H4)2) in 25 mM HEPES/NaOH, pH complex was monitored by native PAGE (Supplementary Fig. 1d). NCPs and Set2 7.5, 2 M NaCl, 1 mM DTT. After an overnight incubation, the histone octamer was were incubated for 1 h on ice in 25 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM DTT, fi 160 μm SAM. Twofold excess of Set over NCPs lead to 50% shift of the NCP band. puri ed from the unincorporated components on the size exclusion Superdex 200 fi Increase 10/300 GL column equilibrated in 25 mM HEPES/NaOH, pH 7.5, 2 M At higher, vefold excess of Set2 over NCPs we have observed the sample pre- NaCl, 1 mM DTT buffer. cipitation. Therefore, for cryo-EM sample preparation we have incubated NCPs fi + with twofold excess of Set using identical conditions as for native PAGE analysis. All protein puri cation steps were done at 4 °C. The protein samples were – analyzed by 17% SDS-PAGE after each purification step (Supplementary Fig. 1b). Three microliters of the sample (1 1.2 mg/ml) were applied to freshly glow- discharged Quantifoil R2/1 holey carbon grid. After 3 s blotting time, grids were plunge-frozen in the liquid ethane using FEI Vitrobot automatic plunge freezer. Humidity in the chamber was kept at 95%. Generation of nonhydrolyzable ubiquitin-H2B histone mimic . The expression Electron micrographs were recorded on FEI Titan Krios at 300 kV with a Gatan plasmid containing a 6xHis-tag at the N-terminus of ubiquitin was a generous gift Summit K2 electron detector (~3350 micrographs) (Necen, Leiden, Netherlands). of Yao T56. Ubiquitin expression was induced in E. coli Rosetta cells with 1 mM fi fi Image pixel size was 1.04 Å per pixel on the object scale. Data were collected in a IPTG at 37 °C for 3 h. Cell lysis and Ni-NTA af nity puri cation under denatur- defocus range of 7000–30,000 Å with a total exposure of 75 e/Å2. Fifty frames were ating conditions were performed as described for Set2 methyltransferase enzyme collected and aligned with the Unblur software package using a dose filter76. except that all buffers contained additional 6 M urea. Eluated protein was Several thousand particles were manually picked and carefully cleaned in Relion exhaustively dialyzed against four changes of water before lyophilization. to remove inconsistent particles. The resulting useful particles were then used for Lyophilized His6-Ub(G76C) and H2B(K120C) were individually resuspended semi-automatic and automatic particle picking in Relion. The contrast transfer in 100 mM sodium tetraborate, pH 8.5, 6 M urea for 1 h at room temperature. function parameters were determined using CTFFIND477. The 2D class averages Solubilized proteins were clarified by centrifugation for 20 min at 17,000 rpm. 78 fi were generated with Relion software package . Inconsistent class averages were TCEP was added to the protein samples at 10 mM nal concentration and removed from further data analysis. The 3D refinements and classifications were incubated at room temperature for 60 min. To remove any free reducing agent, subsequently done in Relion. All final refinements were done in Relion using the proteins were desalted on PD10 column equlibrated in 50 mM sodium tetraborate, auto refine option. The initial reference was filtered to 60 Å in Relion. C1 symmetry pH 8.5, 6 M urea. Ubiquitin and H2B-K120C histone, concentrated to ~10 mg/ml, was applied during refinements for all classes. Particles were split into two datasets were mixed in molar ratio of 2:1 and cross-linked with 1,3-dichloroacetone in N,N fi ′ 56 and re ned independently and the resolution was determined using the 0.143 cut- -dimethylformamide for 60 min on ice . The reaction was stopped with the off (Relion auto refine option). Local resolution was determined with Relion. All addition of 5 mM DTT and incubated for at least 30 min on ice (Supplementary maps were filtered to local resolution using Relion with a B-factor determined by Fig. 7a). Relion. The cross-linking reaction was dialyzed in Ni-NTA denaturing binding buffer Molecular models were built using Coot79 and refined in Phenix80. Electrostatic (50 mM sodium-phosphate, pH 8.0, 500 mM NaCl, 6 M urea, 10 mM imidazole, 3 fi β surface potential calculation and gure was done using Pymol (The Pymol mM -mercaptoethanol) and incubated with Ni Sepharose 6 Fast Flow resin at 4 °C Molecular Graphics System, Version 2.0 Schrodinger, LCC)81. Visualization of all for 30 min. The resin was extensively washed with the binding buffer and 4 column cryo-EM maps was done with Chimera82. volumes of the washing buffer with an increased imidazole (50 mM sodium- phosphate, pH 8.0, 500 mM NaCl, 6 M urea, 20 mM imidazole, 3 mM β- mercaptoethanol). The proteins (his-tagged ubiquitin, his-tagged ubiquitin- Histone methyltransferase assay. In vitro histone methyltransferase assays ubiquitin, his-tagged ubiquitin-H2B) were eluted with the buffer containing 300 (HMT) were performed using 0.5 μg of recombinantly purified C. thermophilum mM imidazole (50 mM sodium-phosphate, pH 8.0, 500 mM NaCl, 6 M urea, 300 Set2 and 2 μg of the nucleosomal substrates assembled with 149 bp DNA along

NATURE COMMUNICATIONS | (2019) 10:3795 | https://doi.org/10.1038/s41467-019-11726-4 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11726-4 with 160 μm SAM, 25 mM Tris/HCl pH 8.4, 50 mM NaCl, 1 mM DTT. For the 16. Carrozza, M. J. et al. Histone H3 methylation by Set2 directs deacetylation of enzymatic reactions HMT buffer was used in the last dialysis step of the nucleo- coding regions by Rpd3S to suppress spurious intragenic transcription. Cell somes assembly reaction. Set2 enzyme was 20 times diluted in HMT buffer to 0.5 123, 581–592 (2005). μg/μl (25 mM Tris/HCl pH 8.4, 50 mM NaCl, 1 mM DTT). The reaction was 17. Li, B. et al. Histone H3 lysine 36 dimethylation (H3K36me2) is sufficient to incubated at 40 °C and at each time point 20 μL aliquots were withdraw and the recruit the Rpd3s histone deacetylase complex and to repress spurious reaction stopped by adding 4xSDS loading dye. The reaction mixtures were ana- transcription. J. Biol. Chem. 284, 7970–7976 (2009). lyzed by SDS-PAGE followed by western blot. Antibodies used include H3K36me1 18. Pai, C.-C. et al. A histone H3K36 chromatin switch coordinates DNA double- (ab9048), H3K36me3 (ab9050), H3 (ab1791), and HRP-conjugated anti-rabbit strand break repair pathway choice. Nat. Commun. 5, 4091 (2014). secondary antibody (Biorad, 170-6515). Membrane was incubated with the primary 19. Carvalho, S. et al. SETD2 is required for DNA double-strand break repair and and the secondary antibody for 1 h at room temperature with the washing steps activation of the p53-mediated checkpoint. Elife 3, e02482 (2014). between each incubation. The signal was visualized with chemoluminescent 20. Jha, D. K., Strahl, B. D. & An, R. N. A. polymerase II-coupled function for substrate. histone H3K36 methylation in checkpoint activation and DSB repair. Nat. 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Wang, Y., Niu, Y. & Li, B. Balancing acts of SRI and an auto-inhibitory appropriate credit to the original author(s) and the source, provide a link to the Creative domain specify Set2 function at transcribed chromatin. Nucleic Acids Res. 43, Commons license, and indicate if changes were made. The images or other third party 4881–4892 (2015). material in this article are included in the article’s Creative Commons license, unless 71. Chesi, M. et al. The t(4;14) translocation in myeloma dysregulates both FGFR3 indicated otherwise in a credit line to the material. If material is not included in the and a novel gene, MMSET, resulting in IgH/MMSET hybrid transcripts. Blood ’ 92, 3025–3034 (1998). article s Creative Commons license and your intended use is not permitted by statutory 72. Ulrich, A., Andersen, K. R. & Schwartz, T. U. 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